PROJECT ABSTRACT Ischemic cardiovascular disease (CVD) is caused by atherosclerosis, a lipid-driven inflammatory disease affecting the arteries, which can progress into vulnerable plaques and thrombotic occlusion. The precise molecular mechanisms linking nutrient excess and hyperlipidemia to immune activation remains elusive and the discovery of these mechanisms could lead to novel CVD therapeutics. An important primer for inflammation in dyslipidemia is the chronic metabolic overloading and impairment of anabolic and catabolic organelles. Reduction of organelle stress alleviates insulin resistance and atherosclerosis. Recently, we showed that small molecule inhibitors of Inositol-requiring enzyme -1 (IRE1), a proximal ER stress sensor, counteract atherosclerosis progression. The ER membranes also serve as a nucleation site for RNA-induced silencing complex (RISC), and we made the striking discovery that IRE1 kinase phosphorylates the double stranded RNA-binding protein, the protein activator of the protein kinase R (PACT), that associates with RISC. We found lipid stress induces IRE1 to phosphorylate PACT, which suppresses mitochondrial biogenesis (mito-biogenesis), in part by controlling a miRNA (miR)-181c. Homeostatic mechanisms such as mitophagy (to remove) or mito-biogenesis (to replenish) the malfunctioning mitochondria can counteract inflammation and also operate in atherosclerotic plaque cells. Aberrant activation of IRE1-PACT signaling by lipids block mito-biogenesis and propagate mitochondrial oxidative (MOX) stress and inflammation, indicating inhibition of this pathological signaling could counteract atherosclerosis. PACT is proximal to a locus on human chromosome 2 that is linked to premature coronary artery disease and plasma HDL-C levels. PACT expression is induced during atherosclerosis progression and reduced during regression in mice. We hypothesize that suppressing IRE1-PACT signaling will promote mito-biogenesis and counteract inflammation and atherosclerosis. We will elucidate how PACT regulates mito-biogenesis by discovering PACT?s miR target(s) and their RNA targets that are relevant to mito-biogenesis regulation. We discovered miR-181c is one of these PACT targets that blocks mito-biogenesis. We will directly investigate the impact of PACT and miR-181c on hyperlipidemia-induced mito-biogenesis, inflammation and atherosclerosis in vivo. Based on the discovered targets (for miR-181c and others) we will develop a more specific therapeutic targeting approach (using Locked Nucleic Acid-Target-Site Blockers) to ablate miR and target interaction in atherosclerotic mice. The successful completion of these studies will help define an unprecedented mechanism of immune-metabolic crosstalk between ER and mitochondria by which hyperlipidemia can promote MOX stress, inflammation and atherosclerosis. Understanding the intrinsic operation of this RNA-mediated inter-organelle communication during atherogenesis could pave the way for novel therapeutic approaches targeting this specific immune-metabolic cross talk in CVD.